# User:Allison K. Alix/Notebook/Thesis Research/2013/06/28

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 Revision as of 10:23, 28 June 2013 (view source) (→Entry title)← Previous diff Current revision (14:36, 12 July 2013) (view source) (9 intermediate revisions not shown.) Line 1: Line 1: {|{{table}} width="800" {|{{table}} width="800" |- |- - |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] Project name + |style="background-color: #EEE"|[[Image:owwnotebook_icon.png|128px]] FCS Measurements |style="background-color: #F2F2F2" align="center"| [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]
{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]      }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]}} |style="background-color: #F2F2F2" align="center"| [[{{#sub:{{FULLPAGENAME}}|0|-11}}|Main project page]]
{{#if:{{#lnpreventry:{{FULLPAGENAME}}}}|[[{{#lnpreventry:{{FULLPAGENAME}}}}{{!}}Previous entry]]      }}{{#if:{{#lnnextentry:{{FULLPAGENAME}}}}|[[{{#lnnextentry:{{FULLPAGENAME}}}}{{!}}Next entry]]}} |- |- | colspan="2"| | colspan="2"| - ==FCS Sample Prep== + ==FCS Measurements== * Run the following samples of MB/DNA * Run the following samples of MB/DNA - 1) 5nM MB 4nM DNA + 1) 10nM MB + 8nM DNA - 2) 1nM MB 960pM DNA + 2) 5nM MB 4nM DNA - 3) 500pM MB 400pM DNA + 3) 1nM MB 960pM DNA - 4) 100pM MB 80pM DNA + 4) 500pM MB 400pM DNA - 5) 50pM MB 40pM MB + 5) 100pM MB 80pM DNA - 6) 25pM MB 20pM DNA + 6) 50pM MB 40pM MB + + 7) 25pM MB 20pM DNA * Run the following oligio-D samples * Run the following oligio-D samples Line 33: Line 35: 5) 100pM 5) 100pM + + ==Calculations== + ''' + MOLECULAR BEACON:''' + + * 7.61μM (x) = 20nM (1000uL) + + x = 2.62uL 7.61uM MB in 997.38uL PBS + + * 20nM (x) = (30uL)(10nM) + + 15uL in 15uL PBS + + * 20nM (x) = 2nM(100uL) + + 10uL in 90uL + + * 2nM (x) = 1nM (100uL) + + x = 50uL in 50uL PBS + + * 200pM (100uL) = x (1nM) + + x = 20uL in 80uL PBS + + * 100pM(100uL) = (200pM)(x) + + x = 50uL in 50uL buffer + + * 50pM (30uL) = 100pM (x) + + x = 15uL in 15uL + + '''DNA:''' + + * 5.31uM (500uL) = x (53.1uM) + + x = 50uL in 450uL PBS + + * 5.31uM (x) = 16nM (1000uL) + + x = 3uL in 997uL PBS + + * 16nM (x) = (8nM) (100uL) + + x = 50uL in 50uL PBS + + * 1.92nM (100uL) = x (8nM) + + x = 24uL in 76 uL PBS + + * 0.8nM (100uL) = x (1.92nM) + + x = 41.7uL in 58.3uL + + * 160pM (100) = x (800pM) + + x = 20uL in 80uL + + * 80pM (100uL) = x (160pM) + + x = 50uL in 50uL + + * 40pM (30uL) = x (80pm) + + x = 15uL in 15uL PBS + + '''OLIGIO-D''' + + * 10uM (x) = 1500uL (20000pM) + + x = 3uL in 1497uL PBS + + * 20000pM (x) = 1000pM (100uL) + + 5 uL in 95 uL PBS + + * 1000pM (x) = 260uL (100pM) + + 26uL in 234uL PBS + + * 80pM = 80uL 100pM in 20uL PBS + + * 60pM = 60uL 100pM in 40uL PBS + + * 40pM = 40uL 100pM in 60uL PBS + + * 20pM = 20uL 100pM in 80uL PBS + + ==FCS Data== + + [[Image:OligioD_calibration_image_OWW.png]] + + The above image is a calibration curve of oligio D samples run for 300s with varying concentrations. We previously saw a trend in the 80pM, 60pM, and 40pM samples. This time, there was a linear trend in the 100pM, 80pM, and 60pM samples with the 40pM and 20pM samples dropping off. This might be an indication of a detection limit, however because we ran these same concentrations before, and the 40pM seemed to follow a linear trend with at least 2 others, we will be running these samples again. We plan to run some slightly higher than 100pM also. + + + + [[Image:MB DNA calibration OWW imageeee.png]] + + The above image shows a calibration curve corresponding to MB/DNA samples. The concentration shown on the graph is the concentration of the MB. The concentration of the DNA was 20% lower to ensure there was excess MB to bind. From this graph, we see a linear trend between the 500pM, 100pM, and 50pM samples with the 25pM sample dropping off. + + + + + +

## Current revision

FCS Measurements Main project page
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## FCS Measurements

• Run the following samples of MB/DNA

1) 10nM MB + 8nM DNA

2) 5nM MB 4nM DNA

3) 1nM MB 960pM DNA

4) 500pM MB 400pM DNA

5) 100pM MB 80pM DNA

6) 50pM MB 40pM MB

7) 25pM MB 20pM DNA

• Run the following oligio-D samples

1) 20pM

2) 40pM

3) 60pM

4) 80pM

5) 100pM

## Calculations

MOLECULAR BEACON:

• 7.61μM (x) = 20nM (1000uL)

x = 2.62uL 7.61uM MB in 997.38uL PBS

• 20nM (x) = (30uL)(10nM)

15uL in 15uL PBS

• 20nM (x) = 2nM(100uL)

10uL in 90uL

• 2nM (x) = 1nM (100uL)

x = 50uL in 50uL PBS

• 200pM (100uL) = x (1nM)

x = 20uL in 80uL PBS

• 100pM(100uL) = (200pM)(x)

x = 50uL in 50uL buffer

• 50pM (30uL) = 100pM (x)

x = 15uL in 15uL

DNA:

• 5.31uM (500uL) = x (53.1uM)

x = 50uL in 450uL PBS

• 5.31uM (x) = 16nM (1000uL)

x = 3uL in 997uL PBS

• 16nM (x) = (8nM) (100uL)

x = 50uL in 50uL PBS

• 1.92nM (100uL) = x (8nM)

x = 24uL in 76 uL PBS

• 0.8nM (100uL) = x (1.92nM)

x = 41.7uL in 58.3uL

• 160pM (100) = x (800pM)

x = 20uL in 80uL

• 80pM (100uL) = x (160pM)

x = 50uL in 50uL

• 40pM (30uL) = x (80pm)

x = 15uL in 15uL PBS

OLIGIO-D

• 10uM (x) = 1500uL (20000pM)

x = 3uL in 1497uL PBS

• 20000pM (x) = 1000pM (100uL)

5 uL in 95 uL PBS

• 1000pM (x) = 260uL (100pM)

26uL in 234uL PBS

• 80pM = 80uL 100pM in 20uL PBS
• 60pM = 60uL 100pM in 40uL PBS
• 40pM = 40uL 100pM in 60uL PBS
• 20pM = 20uL 100pM in 80uL PBS

## FCS Data

The above image is a calibration curve of oligio D samples run for 300s with varying concentrations. We previously saw a trend in the 80pM, 60pM, and 40pM samples. This time, there was a linear trend in the 100pM, 80pM, and 60pM samples with the 40pM and 20pM samples dropping off. This might be an indication of a detection limit, however because we ran these same concentrations before, and the 40pM seemed to follow a linear trend with at least 2 others, we will be running these samples again. We plan to run some slightly higher than 100pM also.

The above image shows a calibration curve corresponding to MB/DNA samples. The concentration shown on the graph is the concentration of the MB. The concentration of the DNA was 20% lower to ensure there was excess MB to bind. From this graph, we see a linear trend between the 500pM, 100pM, and 50pM samples with the 25pM sample dropping off.